The aim of this application is to develop an improved vaccine for protection against smallpox. This vaccine will be based on attenuating mutations that we have identified in vaccinia virus. These mutations decrease neurovirulence of vaccinia virus 1,000 to >100,000-fold, but still provide excellent protection against challenge with a pathogenic poxvirus. These viruses are also reduced for pathogenesis in immune-compromised mice. Since encephalitis and disseminated disease in immune-compromised humans are amongst the most severe complications to use of vaccinia virus as a vaccine, these mutants should be safer to use than the currently available strains of vaccinia virus. We are proposing to insert these mutations into a strain of vaccinia virus that is appropriate for use in humans (NYCBH), under conditions that will allow use of these viruses in humans. Strains will be analyzed in mice for pathogenesis and for induction of a protective immune response.